Transglutaminases (TGases) catalyze the formation of a cross-link between a donor amide group of a protein-bound glutamine residue and an acceptor epsilon-NH2 of a protein-bound lysine residue. This cross-link is an isopeptide bond that cannot be cleaved in vertebrate organisms. The net result therefore is the formation of a permanent, stable, insoluble macromolecular protein complex. In the epidermis and other stratified squamous epithelia, several of the nine known TGase enzymes are expressed. In particular, TGases 1, 2 and 3 cross-link a variety of defined structural proteins to form the cornified cell envelope which is a principal component of epithelial barrier function. We are studying in detail each of these enzymes, and their roles in diseases. Transglutaminase 1 The TGase 1 enzyme in cultured keratinocytes or foreskin epidermal cells is complex since it exists in multiple soluble and membrane-bound full-length as well as proteolytically-processed forms. Most of the enzyme is membrane bound by way of myristate and anchorages on the amino-terminal segment which is unique to the TGase 1 enzyme. The various forms display wide variations in specific activities, but these are difficult to measure because the enzyme is inherently unstable and easily degraded by proteolysis. To address structural and functional questions, we have been successful in expression in baculovirus systems. Previous work from this laboratory has shown that mutations in the TGM1 gene, encoding the TGase 1 enzyme, cause the autosomal recessive disorder lamellar ichthyosis. We have expressed in baculovirus several of the known mutations to explore the bases of loss of enzyme activity. These studies have been coupled with structural analyses based on comparative structure with the TGase 3 and factor XIIIa enzymes. In this way, we hope to gain a better understanding of the role of this enzyme in the skin. These studies offer an opportunity to understand its structure and substrate specificity. Transglutaminase 2 Our main focus of this enzyme is to obtain atomic resolution structural information by X-ray diffraction of crystals. To date, we have developed methods for the large scale preparation and purification of the active enzyme in baculovirus. Ongoing work will involve crystallization trials. Transglutaminase 3 The TGase 3 enzyme is expressed in many epithelial cell types, initially as an inactive pro-enzyme, that requires proteolytic activation by specific cleavage. In addition, data from this laboratory have shown that it is the preferred enzyme for cross-linking in vivo of several important substrates involved in barrier or other functions, including loricrin, small proline rich proteins, and trichohyalin. We have developed methods for the large-scale expression and purification of several forms of the TGase 3 enzyme in the baculovirus system. These include the pro-enzyme, activated enzyme, and the 50 kDa active form. Each of these has been crystallized and the structures have been resolved by X-ray diffraction methods to 2.5 A or less. For example, the activated form acquired 3 calcium ions that are required for activity and which cause a significant change in structure as compared to the zymogen. These data have provided the basis for a new understanding of the TGase enzyme reaction mechanism. Several residues have identified at or near the active site region that may confer substrate specificity for the enzyme. These studies will continue. Hopefully, they will allow us to rationally design specific inhibitors for this enzyme. Expression of TGases in non-epithelial tissues: roles in degenerative diseases By RT-PCR methods, we have found that TGases 1, 2 and 3 are widely expressed in a variety of non-epithelial tissues, including in particular, various tissues within the brain, connective tissues, fibroblasts and muscle. Heretofore, these tissues were thought to express only the cytosolic TGase 2 enzyme. The expression of these enzymes has been confirmed by both immunoprecipitation and indirect immunofluorescence methods using specific probes. Moreover, mRNA and enzyme levels of TGases 1 and 3 are up-regulated in pathological conditions, including Alzheimers Disease (AD), sporadic inclusion body myositis (SIBM) and celiac disease. Together, these data demonstrate that elevated levels of TGases 1 and/or 3 correlate with disease pathogenesis and contribute directly to the formation of insoluble cross-linked bodies that interfere with normal cellular function and thus degenerative disease. Depending on availability of adequate amounts of tissue, further work will be directed toward analysis of the forms of the TGase 1 and 3 enzymes and more detailed sequencing analyses. We are performing parallel studies with other diseases including celiac disease, type II diabetes and Huntingtons Disease, all of which have aberrantly high levels of TGase expression. New members of the TGase family Three new members of the TGase family have been discovered recently, and at least two of these, TGases 5 and 6, are abundantly expressed in epithelia. We have acquired expression vectors for both and will express these with the aim of performing additional structural studies. Further, we will explore their roles in diseases.